Mechanism of Action of Interferon in the Treatment of Myeloproliferative Neoplasms

Mechanism of Action of Interferon in the Treatment of Myeloproliferative Neoplasms

Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include: Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). They are myeloid malignancies resulting from the transformation of a multipotent hematopoietic stem cell (HSC) caused by mutations activating the JAK2/STAT pathway. The most prevalent mutation is JAK2V617F. Type 1 and Type 2 calreticulin (CALR) and thrombopoietin receptor (MPL) mutations are also observed in ET and PMF. Additional non-MPN mutations affecting different pathways are also found, particularly in PMF, and are involved in disease initiation and/or in phenotypic changes and /or disease progression and/or response to therapy.

There is an obvious and urgent need for an efficient therapy for MPN. In particular, PMF remain without curative treatment, except allogeneic HSC transplantation and JAK inhibitors have limited effects on the disease outcome. Among novel therapeutic approaches, Peg-IFNα2a (IFN) is the most efficient harboring both high rates of hematological responses in JAK2V617F and CALRmut MPN patients and some molecular responses mainly in JAK2V617F patients including deep molecular response (DMR). Nevertheless, several studies, including our own, have demonstrated that the IFN molecular response in CALRmut patients is heterogeneous and overall much lower than in JAK2V617F patients. Moreover, some JAK2V617F MPN patients do not respond to IFN, and DMR is only observed in around 20% of JAK2V617F patients. Finally, long-term treatments are needed (2-5 years) to obtain a DMR, jeopardizing its success due to possible long-term toxicity.

The underlying reasons for failure, drug resistance, heterogeneous molecular response in CALRmut patients and the long delays for DMR in JAK2V617F patients remain unclear, largely because the mechanisms by which IFNα targets MPN malignant clones remain elusive.

Significant improvement of IFN efficacy cannot be achieved without basic and clinical research. Hence our two lines of research are to

• Understand how IFNα specifically targets neoplastic HSCs
• Predicting and improving patient response during IFNα therapy

Classical BCR-ABL-negative myeloproliferative neoplasms (MPN) include: Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). They are myeloid malignancies resulting from the transformation of a multipotent hematopoietic stem cell (HSC) caused by mutations activating the JAK2/STAT pathway. The most prevalent mutation is JAK2V617F. Type 1 and Type 2 calreticulin (CALR) and thrombopoietin receptor (MPL) mutations are also observed in ET and PMF. Additional non-MPN mutations affecting different pathways are also found, particularly in PMF, and are involved in disease initiation and/or in phenotypic changes and /or disease progression and/or response to therapy.

There is an obvious and urgent need for an efficient therapy for MPN. In particular, PMF remain without curative treatment, except allogeneic HSC transplantation and JAK inhibitors have limited effects on the disease outcome. Among novel therapeutic approaches, Peg-IFNα2a (IFN) is the most efficient harboring both high rates of hematological responses in JAK2V617F and CALRmut MPN patients and some molecular responses mainly in JAK2V617F patients including deep molecular response (DMR). Nevertheless, several studies, including our own, have demonstrated that the IFN molecular response in CALRmut patients is heterogeneous and overall much lower than in JAK2V617F patients. Moreover, some JAK2V617F MPN patients do not respond to IFN, and DMR is only observed in around 20% of JAK2V617F patients. Finally, long-term treatments are needed (2-5 years) to obtain a DMR, jeopardizing its success due to possible long-term toxicity.

The underlying reasons for failure, drug resistance, heterogeneous molecular response in CALRmut patients and the long delays for DMR in JAK2V617F patients remain unclear, largely because the mechanisms by which IFNα targets MPN malignant clones remain elusive.

Significant improvement of IFN efficacy cannot be achieved without basic and clinical research. Hence our two lines of research are to

• Understand how IFNα specifically targets neoplastic HSCs
• Predicting and improving patient response during IFNα therapy

The main objective from the basic point of view is to draw the clonal architecture of the mutated cells of the patients during IFN treatment to provide a better understanding of the mechanism of action of IFN in MPN: namely how and at what level of hematopoietic differentiation the IFN specifically targets JAK2V617F HSCs and if and why it does not have the same effect on CALRm patients.

Our previous clinical study using clonal architecture data combined with a mathematical model indicates that depletion of JAK2V617F HSC by differentiation into progenitors and thus loss of self-renewal may be the critical mechanism for eradication of JAK2V617F disease by IFN. We hope to confirm this hypothesis in a larger number of patients and to understand the basis of the differential effects of JAK2V617F and CALRm mutations on disease-initiating stem cells.

The secondary objectives are to:

• Validate the resistance of CALRm patients to IFN treatment in a larger number of patients. Moreover, high IFN doses, in contrast to JAK2V617F patients, are deleterious to the molecular response for reasons that remain to be understood.
• Investigate the role of associated mutations in IFN-induced molecular responses.

IFN treatments have been shown to promote the appearance of clones (JAK2V617F positive or JAK2V617F negative) with additional mutations, such as Tet2 or DNMT3a, which could be responsible for resistance to IFN treatment . We would like to increase the number of patients and their follow-up to analyze the role of these mutations in treatment success. Moreover, these additional mutations (new or selected by the treatment) could favor the development of more severe pathologies (MF, MDS, AML) than PV or TE and would be important to monitor on the follow-up of IFN-treated patients.

-Explore in vitro the effect of IFN in combination with other molecules on primary patients' cells. Indeed, our basic study already showed the involvement of PML in the mechanism of action of IFN and we found that arsenic greatly potentiates the effect of IFN. We will deeply investigate by which exact mechanisms.

All the data will be collected in patients before IFN treatment or during the IFN treatment and data will be collected by single cell genotyping of colonies and/or by single cell RNA sequencing coupled to genotyping of mutations and/or in vitro assays.

Inclusion Criteria:

1. Adult male or female 18 years of age or older
2. Diagnosis of MPN has been previously established by the referring physician and that physician will have decided to treat with pegylated IFN. Patients will be treated or untreated at the time of inclusion and may be newly diagnosed patients.
3. These patients will be affiliated with or benefit from a social security plan
4. For all these patients an additional 20-40 mL will be collected except for some PV patients who are treated conventionally by phlebotomy. In this case, we will collect blood bags from these patients. The volumes vary between 300 and 450 mL of blood depending on the weight and size of the patients.
5. We will also include in this protocol any patient whose MPN, either PV, TE or MF, will have progressed to acute leukemia (AL) during treatment. These will be patients with AP of MPN (MPN can also progress to acute myeloid leukemia (AML) by acute transformation (AT) of MPN).
6. Patient with signed informed consent

Exclusion Criteria:

1. The non-inclusion criterion concerns the anemia that some MF patients may suffer from. Therefore, patients with anemia (Hb<10g) or transfusion dependency (≥ 1 packed red blood cell per month) at the time of the referral monitoring visit are not included in the research.
2. Persons under court protection, guardianship or curatorship